Embedding resin composition for electron microscope, and method for observing sample on electron microscope using said composition

ABSTRACT

The present invention provides an embedding resin composition for electron microscopy having satisfactory performance as an embedding medium, including embedding performance and sectioning quality, and exhibiting excellent antistatic performance; and a method for observing a sample with an electron microscope using the composition. The embedding resin composition for electron microscopy of the present invention having antistatic performance comprises an ionic liquid and an embedding medium comprising an epoxy-based resin, a methacrylate resin or an unsaturated polyester resin. Preferably, the ionic liquid comprising
         a quaternary ammonium compound based on the formula (I):       

     
       
         
         
             
             
         
       
     
     and
         an anion selected from the group consisting of BF 4   − , PF 6   − , (CF 3 SO 2 ) 2 N − , a halide ion, a conjugate base of carboxylic acid, a conjugate base of sulfonic acid and a conjugate base of an inorganic acid.

TECHNICAL FIELD

The present invention relates to an embedding resin composition forelectron microscopy and a method for observing a sample with an electronmicroscope using the composition.

BACKGROUND ART

Electron microscopes are generally used to investigate theultrastructure of biological samples such as cells and tissue, polymerresin samples, and crystalline samples such as inorganic substances. Twotypes of electron microscopes are known, including scanning electronmicroscopes (hereinafter sometimes referred to as SEMs) and transmissionelectron microscopes (hereinafter sometimes referred to as TEMs).

Patent Literature 1 describes a sample preparation method in which asample to be observed is embedded in an embedding medium and thinsectioned with a microtome etc. The embedding medium used is an epoxyresin, an unsaturated polyester resin, an acrylic resin, or the like,all of which are insulating materials. Consequently, the resin tends tocharge at the time of observation with an electron microscope and thismay cause difficulty in examining the subject. In some cases suchcharging may result in discharge phenomena inside the electronmicroscope, which may damage the detectors. These problems becomeobstacles especially when insulating materials are observed with a SEM.

In order to overcome the problems relating to charging duringobservation with a SEM, various modifications of observation techniquehave been conventionally introduced. For example, observation with a SEMhas been conventionally carried out under conditions in which incidentelectrons and emitted electrons are in a particular angle relationship,or under low-vacuum conditions, or under low-current conditions.However, these modifications have drawbacks. For example, theobservation range is narrow, contrast and resolution are insufficient,and elemental analysis cannot be conducted.

Patent Literature 2 discloses an epoxy resin composition havingantistatic performance, produced by adding an alkali metal salt and apolyether-based polymer to an epoxy resin. However, the compositionlacks sufficient mechanical strength and therefore does not providesatisfactory performance as an embedding medium, including embeddingperformance and sectioning quality.

CITATION LIST Patent Literature

-   Patent Literature 1: JP 5-26794 A-   Patent Literature 2: JP 3-122165 A

SUMMARY OF INVENTION Technical Problem

The present invention was developed in view of the above problems in theconventional art. An object of the present invention is to provide anembedding resin composition for electron microscopy having satisfactoryperformance as an embedding medium, including embedding performance andsectioning quality, and exhibiting excellent antistatic performance; andto provide a method for observing a sample with an electron microscopeusing the composition.

Solution to Problem

To solve the above problems, the present invention includes thefollowing.

[1] An embedding resin composition for electron microscopy, thecomposition having antistatic performance and comprising an ionic liquidand an embedding medium comprising an epoxy-based resin, a methacrylateresin or an unsaturated polyester resin.[2] The composition according to the above [1], wherein the ionic liquidcomprising

a quaternary ammonium compound based on the formula (I):

(in the formula (I), R¹, R², R³ and R⁴ are independently a hydrogenatom, an alkyl group, an alkynyl group, an alkenyl group, an alkadienegroup, an alkatriene group, a cycloalkyl group or an aliphaticheterocyclic group and any of the hydrogen atoms of the groups may bereplaced with a substituent; at least one of R¹, R², R³ and R⁴ containsan alkenyl group, an alkadiene group, an alkatriene group or an epoxygroup; and R¹, R², R³ and R⁴ may bind to each other to form a ring) and

an anion selected from the group consisting of BF₄ ⁻, PF₆ ⁻,(CF₃SO₂)₂N⁻, a halide ion, a conjugate base of carboxylic acid, aconjugate base of sulfonic acid and a conjugate base of an inorganicacid.

[3] The composition according to the above [2], wherein R¹ is an epoxygroup, a glycidyl group, or an alkenyl group of 2 to 10 carbon atoms.[4] The composition according to the above [2] or [3], wherein R², R³and R⁴ are independently an alkyl group of 1 to 6 carbon atoms.[5] The composition according to any of the above [2] to [4], whereinthe quaternary ammonium compound is a monomer, an oligomer or a mixturethereof.[6] The composition according to any of the above [2] to [5], whereinthe anion is (CF₃SO₂)₂N⁻.[7] The composition according to any of the above [1] to [6], whereinthe amount of the ionic liquid is 5 to 35 vol % relative to the totalvolume of the composition.[8] A method for observing a sample with an electron microscope, themethod comprising the step of embedding a sample in the compositionaccording to any of the above [1] to [7].[9] The observation method according to the above [8], wherein themethod comprises the step of staining a sample and the staining step isperformed before the embedding step.[10] The observation method according to the above [8] or [9], whereinthe electron microscope is a scanning electron microscope.[11] The observation method according to any of the above [8] to [10],wherein the sample is a biological sample.[12] The observation method according to the above [11], wherein thebiological sample is a cell or tissue of a plant or animal.[13] The observation method according to any of the above [8] to [10],wherein the sample is a non-biological sample.[14] The observation method according to the above [13], wherein thenon-biological sample is a resin, a rubber, a synthetic resin, apigment, a coating material, a cosmetic, a pharmaceutical drug, aceramic, a magnetic body, a magnetic material, a semiconductor, a metal,a metal oxide, a mineral, an organic salt, or an inorganic salt.[15] The observation method according to any of the above [8] to [14],wherein the sample is a powder.

Advantageous Effects of Invention

The embedding resin composition for electron microscopy of the presentinvention has satisfactory performance as an embedding medium, includingembedding performance and sectioning quality, and exhibits excellentantistatic performance.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph showing the results of electron microscopeobservation of hepatocytes embedded in the embedding resin compositionfor electron microscopy of Example 1 (2000-fold magnification).

FIG. 2 is a photograph showing the results of electron microscopeobservation of hepatocytes embedded in the embedding resin compositionfor electron microscopy of Comparative Example 1 (2024-foldmagnification).

FIG. 3 is a photograph showing an electron microscope secondary electronimage of a waste toner embedded in the embedding resin composition forelectron microscopy of Example 4 (200-fold magnification).

FIG. 4 is a photograph showing an electron microscope backscatteredelectron image of a waste toner embedded in the embedding resincomposition for electron microscopy of Example 4 (2000-foldmagnification).

FIG. 5 is a photograph showing an electron microscope secondary electronimage of a waste toner embedded in the embedding resin composition forelectron microscopy of Comparative Example 2 (200-fold magnification).

FIG. 6 is a photograph showing an electron microscope backscatteredelectron image of a waste toner embedded in the embedding resincomposition for electron microscopy of Comparative Example 2 (2000-foldmagnification).

FIG. 7 is a photograph showing an electron microscope secondary electronimage of a plastic sticky note embedded in the embedding resincomposition for electron microscopy of Example 5 (200-foldmagnification).

FIG. 8 is a photograph showing an electron microscope backscatteredelectron image of a plastic sticky note embedded in the embedding resincomposition for electron microscopy of Example 5 (1200-foldmagnification).

FIG. 9 is a photograph showing a backscattered electron image of theadhesive part of a plastic sticky note embedded in the embedding resincomposition for electron microscopy of Example 5 (5000-foldmagnification).

FIG. 10 is a photograph showing an electron microscope secondaryelectron image of a plastic sticky note embedded in the embedding resincomposition for electron microscopy of Comparative Example 3 (200-foldmagnification).

FIG. 11 is a photograph showing an electron microscope backscatteredelectron image of a plastic sticky note embedded in the embedding resincomposition for electron microscopy of Comparative Example 3 (1200-foldmagnification).

FIG. 12 is a photograph showing a three-dimensional reconstruction imageof a waste toner in Example 6.

DESCRIPTION OF EMBODIMENTS

The embedding resin composition for electron microscopy of the presentinvention has antistatic performance and comprises an ionic liquid andan embedding medium comprising an epoxy-based resin, a methacrylateresin or an unsaturated polyester resin.

Ionic Liquid

The ionic liquid used in the present invention refers to a salt which isin the liquid state at normal temperature.

Cation

The cation of the ionic liquid of the present invention is notparticularly limited as long as it constitutes the ionic liquid. Thecation may be, for example, imidazolium, pyridium, pyrrolidinium,quaternary phosphonium, quaternary ammonium, or the like. The cation maybe one kind or a mixture of two or more kinds.

Examples of the imidazolium include 1,3-dimethylimidazolium,1-ethyl-3-methylimidazolium, 1-ethyl-3-methylimidazolium,1-methyl-3-propylimidazolium, 1-butyl-3-methylimidazolium,1-hexyl-3-methylimidazolium, 1-butyl-2,3-dimethylimidazolium, etc.

Examples of the pyridinium include 1-ethylpyridinium, 1-butylpyridinium,1-butyl-4-methylpyridinium, 1-ethyl-3-methylpyridinium,1-ethyl-3-(hydroxymethyl)pyridinium, etc.

Examples of the pyrrolidinium include N-methyl-N-propylpyrrolidinium,N-methyl-N-butylpyrrolidinium, etc.

Examples of the quaternary phosphonium includetributyllaurylphosphonium, tributylmyristylphosphonium,tributylcetylphosphonium, tributylstearylphosphonium,triphenyllaurylphosphonium, triphenylmyristylphosphonium,triphenylcetylphosphonium, triphenylstearylphosphonium,benzyldimethyllaurylphosphonium, benzyldimethylmyristylphosphonium,benzyldimethylcetylphosphonium, benzyldimethylstearylphosphonium, etc.

Examples of the quaternary ammonium include tetraalkylammonium such astetramethylammonium, tetraethylammonium, and triethylmethylammonium;triazolium, pyridazinium, thiazolium, oxazolium, pyrimidinium,pyrazinium, etc.

Preferably, the cation of the ionic liquid used in the present inventionis quaternary ammonium. More preferably, the ionic liquid comprises, asa cation, a quaternary ammonium compound based on the following formula(I):

(in the formula (I), R¹, R², R³ and R⁴ are independently a hydrogenatom, an alkyl group, an alkynyl group, an alkenyl group, an alkadienegroup, an alkatriene group, a cycloalkyl group or an aliphaticheterocyclic group and any of the hydrogen atoms of the groups may bereplaced with a substituent; at least one of R¹, R², R³ and R⁴ containsan alkenyl group, an alkadiene group, an alkatriene group or an epoxygroup; and R¹, R², R³ and R⁴ may bind to each other to form a ring).

The alkyl group represented by R¹, R², R³ and/or R⁴ may be linear orbranched and examples thereof include a linear or branched alkyl groupof 1 to 20 carbon atoms. Specific examples thereof include a linear orbranched alkyl group such as methyl, ethyl, n-propyl, isopropyl,n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl,tert-pentyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, hexyl, isohexyl,4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl,3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl,1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1-ethylbutyl,2-ethylbutyl, heptyl, octyl, nonyl, decyl, cetyl, stearyl, etc.Preferably, the alkyl group is of 1 to 12 carbon atoms, more preferably1 to 6 carbon atoms.

The alkynyl group represented by R¹, R², R³ and/or R⁴ may be linear orbranched and examples thereof include an alkynyl group of 2 to 15 carbonatoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbonatoms. Specific examples thereof include ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 3-butynyl, pentynyl, hexynyl, etc.

The alkenyl group represented by R¹, R², R³ and/or R⁴ may be linear orbranched and examples thereof include an alkenyl group of 2 to 15 carbonatoms, preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbonatoms. Specific examples thereof include vinyl, 1-propenyl, allyl,1-butenyl, 2-butenyl, 3-butenyl, pentenyl, hexenyl, isopropenyl,2-methyl-2-propenyl, 1-methyl-2-propenyl, 2-methyl-1-propenyl, etc.

The alkadiene group represented by R¹, R², R³ and/or R⁴ may be linear orbranched and examples thereof include an alkadiene group of 3 to 15carbon atoms, preferably 4 to 10 carbon atoms. Specific examples thereofinclude butadiene, pentadiene, hexadiene, etc.

The alkatriene group represented by R¹, R², R³ and/or R⁴ may be linearor branched and examples thereof include an alkatriene group of 4 to 15carbon atoms, preferably 6 to 12 carbon atoms. Specific examples thereofinclude hexatriene, heptatriene, octatriene, etc.

The cycloalkyl group represented by R¹, R², R³ and/or R⁴ is, forexample, a cycloalkyl group of 3 to 14 carbon atoms, preferably 5 to 12carbon atoms, more preferably 6 to 12 carbon atoms. Specific examplesthereof include cyclopropyl, cyclobutyl, cyclopentyl, methylcyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, 1,2-dimethylcyclopentyl,1,3-dimethylcyclopentyl, 1-ethyl-2-methylcyclopentyl, etc.

The aliphatic heterocyclic group represented by R¹, R², R³ and/or R⁴ is,for example, a 5- to 8-membered, preferably 5- or 6-membered, monocyclicaliphatic heterocyclic group or a polycyclic or fused aliphaticheterocyclic group, the group having 2 to 14 carbon atoms and containingat least one heteroatom, preferably 1 to 3 heteroatoms, such as anitrogen atom, an oxygen atom and a sulfur atom. Examples of thealiphatic heterocyclic group include pyrrolidyl-2-one, piperidino,piperazinyl, morpholino, tetrahydrofuryl, tetrahydropyranyl,tetrahydrothienyl, etc.

Any of the hydrogen atoms of the alkyl group, the alkynyl group, thealkenyl group, the alkadiene group, the alkatriene group, the cycloalkylgroup or the aliphatic heterocyclic group represented by R¹, R², R³and/or R⁴ may be replaced with a substituent. The substituent is notparticularly limited and examples thereof include alkyl, alkynyl,alkenyl, alkadiene, alkatriene, aryl, alkoxy, alkylenedioxy, aryloxy,aralkyloxy, heteroaryloxy, alkylthio, cycloalkyl, aliphaticheterocyclic, arylthio, aralkylthio, heteroarylthio, amino, substitutedamino, cyano, hydroxyl, oxo, epoxy, glycidyl, nitro, and mercaptogroups, a halogen atom, etc. The number of the substituents ispreferably 1 to 3, more preferably 1 or 2.

The alkyl group as the substituent may be linear or branched andexamples thereof include a linear or branched alkyl group of 1 to 20carbon atoms. Specific examples thereof are the same as those listed forthe above alkyl group represented by R¹, R², R³ and/or R⁴.

The alkynyl group as the substituent may be linear or branched andexamples thereof include an alkynyl group of 2 to carbon atoms,preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms.Specific examples thereof are the same as those listed for the abovealkynyl group represented by R¹, R², R³ and/or R⁴.

The alkenyl group as the substituent may be linear or branched andexamples thereof include an alkenyl group of 2 to carbon atoms,preferably 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms.Specific examples thereof are the same as those listed for the abovealkenyl group represented by R¹, R², R³ and/or R⁴.

The alkadiene group as the substituent may be linear or branched andexamples thereof include an alkadiene group of 3 to 15 carbon atoms,preferably 4 to 10 carbon atoms. Specific examples thereof are the sameas those listed for the above alkadiene group represented by R¹, R², R³and/or R⁴.

The alkatriene group as the substituent may be linear or branched andexamples thereof include an alkatriene group of 4 to 15 carbon atoms,preferably 6 to 12 carbon atoms. Specific examples thereof are the sameas those listed for the above alkatriene group represented by R¹, R², R³and/or R⁴.

The cycloalkyl group as the substituent is, for example, a cycloalkylgroup of 3 to 14 carbon atoms, preferably 5 to 12 carbon atoms, morepreferably 6 to 12 carbon atoms. Specific examples thereof are the sameas those listed for the above cycloalkyl group represented by R¹, R², R³and/or R⁴.

The aliphatic heterocyclic group as the substituent is, for example, a5- to 8-membered, preferably 5- or 6-membered, monocyclic aliphaticheterocyclic group or a polycyclic or fused aliphatic heterocyclicgroup, the group having 2 to 14 carbon atoms and containing at least oneheteroatom, preferably 1 to 3 heteroatoms, such as a nitrogen atom, anoxygen atom and a sulfur atom. Specific examples thereof are the same asthose listed for the above aliphatic heterocyclic group represented byR¹, R², R³ and/or R⁴.

Examples of the aryl group as the substituent include an aryl group of 6to 20 carbon atoms and specific examples thereof include phenyl,1-naphthyl, 2-naphthyl, anthryl, phenanthryl, 2-biphenyl, 3-biphenyl,4-biphenyl, terphenyl, etc.

Examples of the aryloxy group as the substituent include an aryloxygroup of 6 to 14 carbon atoms and specific examples thereof includephenoxy, tolyloxy, xylyloxy, naphthoxy, anthryloxy, etc.

Examples of the aralkyl group as the substituent include a group derivedfrom the above alkyl group by replacing at least one hydrogen atom withthe above aryl group and example thereof include an aralkyl group of 7to 18 carbon atoms. Specific examples thereof include benzyl, phenethyl,1-phenylpropyl, 3-naphthylpropyl, diphenylmethyl, 1-naphthylmethyl,2-naphthylmethyl, 2,2-diphenylethyl, 3-phenylpropyl, 4-phenylbutyl,5-phenylpentyl, etc.

Examples of the aromatic heterocyclic group as the substituent include a5- to 8-membered, preferably 5- or 6-membered, monocyclic heteroarylgroup or a polycyclic or fused heteroaryl group, the group having 2 to15 carbon atoms and containing at least one heteroatom, preferably 1 to3 heteroatoms, such as a nitrogen atom, an oxygen atom and a sulfuratom. Examples of the 5- or 6-membered monocyclic heteroaryl group andthe polycyclic or fused heteroaryl group include furyl, thienyl,pyrrolyl, pyridyl, pyrimidyl, pyrazyl, pyridazyl, pyrazolyl, imidazolyl,oxazolyl, thiazolyl, benzofuryl, benzothienyl, quinolyl, isoquinolyl,quinoxalyl, phthalazyl, quinazolyl, naphthyridyl, cinnolyl,benzimidazolyl, benzoxazolyl, benzothiazolyl, etc.

The alkoxy group as the substituent may be linear, branched or cyclicand examples thereof include an alkoxy group of 1 to 6 carbon atoms.Specific examples thereof include methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, n-pentyloxy,2-methylbutoxy, 3-methylbutoxy, 2,2-dimethylpropyloxy, n-hexyloxy,2-methylpentyloxy, 3-methylpentyloxy, 4-methylpentyloxy,5-methylpentyloxy, cyclohexyloxy, methoxymethoxy, 2-ethoxyethoxy, etc.

Examples of the alkylenedioxy group as the substituent include analkylenedioxy group of 1 to 3 carbon atoms. Specific examples thereofinclude methylenedioxy, ethylenedioxy, trimethylenedioxy,propylenedioxy, isopropylidenedioxy, etc.

The alkylthio group as the substituent may be linear, branched or cyclicand examples thereof include an alkylthio group of 1 to 6 carbon atoms.Specific examples thereof include methylthio, ethylthio, n-propylthio,isopropylthio, n-butylthio, 2-butylthio, isobutylthio, tert-butylthio,pentylthio, hexylthio, cyclohexylthio, etc.

Examples of the aralkyloxy group as the substituent include anaralkyloxy group of 7 to 12 carbon atoms and specific examples thereofinclude benzyloxy, 1-phenylethoxy, 2-phenylethoxy, 1-phenylpropoxy,2-phenylpropoxy, 3-phenylpropoxy, 1-phenylpentyloxy, 2-phenylpentyloxy,3-phenylpentyloxy, 4-phenylpentyloxy, 5-phenylpentyloxy,1-phenylhexyloxy, 2-phenylhexyloxy, 3-phenylhexyloxy, 4-phenylhexyloxy,5-phenylhexyloxy, 6-phenylhexyloxy, etc.

Examples of the arylthio group as the substituent include an arylthiogroup of 6 to 14 carbon atoms and specific examples thereof includephenylthio, tolylthio, xylylthio, naphthylthio, etc.

Examples of the heteroaryloxy group as the substituent include aheteroaryloxy group having 2 to 14 carbon atoms and containing at leastone heteroatom, preferably 1 to 3 heteroatoms, such as a nitrogen atom,an oxygen atom, and a sulfur atom. Specific examples thereof include2-pyridyloxy, 2-pyrazyloxy, 2-pyrimidyloxy, 2-quinolyloxy, etc.

Examples of the aralkylthio group as the substituent include anaralkylthio group of 7 to 12 carbon atoms and specific examples thereofinclude benzylthio, 2-phenethylthio, etc.

Examples of the heteroarylthio group include a heteroarylthio grouphaving 2 to 14 carbon atoms and containing at least one heteroatom,preferably 1 to 3 heteroatoms, such as a nitrogen atom, an oxygen atomand a sulfur atom. Specific examples thereof include 2-pyridylthio,4-pyridylthio, 2-benzimidazolylthio, 2-benzoxazolylthio,2-benzthiazolylthio, etc.

Examples of the substituted amino group include a group derived from anamino group by replacing one or two hydrogen atoms with a substituentsuch as an alkyl group, an aryl group and an aralkyl group.

Examples of the amino group substituted with an alkyl group, i.e., thealkyl-substituted amino group include a mono- or dialkylamino group suchas N-methylamino, N,N-dimethylamino, N,N-diethylamino,N,N-diisopropylamino, N-cyclohexylamino, etc.

Examples of the amino group substituted with an aryl group, i.e., thearyl-substituted amino group include a mono- or diarylamino group suchas N-phenylamino, N,N-diphenylamino, N,N-ditolylamino, N-naphthylamino,N-naphthyl-N-phenylamino, etc.

Examples of the amino group substituted with an aralkyl group, i.e., thearalkyl-substituted amino group include a mono- or diaralkylamino groupsuch as N-benzylamino, N,N-dibenzylamino, etc.

One or more hydrogen atoms of the above substituents may be replacedwith a halogen atom. Examples of the halogen atom include a fluorineatom, a chlorine atom, a bromine atom, an iodine atom, etc.

In the formula (I), at least one of R¹, R², R³ and R⁴ contains analkenyl group, an alkadiene group, an alkatriene group or an epoxygroup.

Examples of such a group containing an epoxy group include an epoxygroup, a glycidyl group, etc., and preferred is a glycidyl group in viewof reactivity.

Examples of the alkenyl group, the alkadiene group and the alkatrienegroup are the same as those listed above for R¹, R², R³ and R⁴.

R¹, R², R³ and R⁴ may bind to each other to form a ring. The ring is,for example, a 3- to 10-membered, preferably 5- or 6-membered,monocyclic ring or a polycyclic ring and may contain a heteroatom suchas a nitrogen atom, an oxygen atom and a sulfur atom. Any two of, orthree or more of R¹, R², R³ and R⁴ may bind to each other to form aring. Examples of the ring include methylcyclopentyl, cyclohexyl, furyl,thienyl, pyrrolyl, pyridyl, etc.

Preferably, R¹ is an epoxy group, a glycidyl group, or an alkenyl groupof 2 to 10 carbon atoms. This is because an epoxy group, a glycidylgroup and an alkenyl group of 2 to 10 carbon atoms are highly reactiveand therefore exhibit excellent antistatic performance when mixed withan embedding medium.

Examples of the alkenyl group of 2 to 10 carbon atoms represented by R¹include vinyl, 1-propenyl, allyl, 1-butenyl, 2-butenyl, 3-butenyl,pentenyl, hexenyl, isopropenyl, 2-methyl-2-propenyl,1-methyl-2-propenyl, 2-methyl-1-propenyl, etc.

Particularly preferably, R¹ is a glycidyl group or an alkenyl group of 1to 6 carbon atoms in view of reactivity.

Preferably, R², R³ and R⁴ are independently an alkyl group of 1 to 6carbon atoms, more preferably an alkyl group of 1 to 4 carbon atoms,particularly preferably a methyl group or an ethyl group. When R², R³and R⁴ are the above groups, steric hindrance on R¹ can be prevented andsufficient reactivity can be obtained.

Preferably, the quaternary ammonium compound is a monomer, an oligomeror a mixture thereof. Examples of the quaternary ammonium compound as anoligomer include a quaternary ammonium compound in a dimer or trimerform or in the form of an oligomer with a molecular weight of about 1000or less. Examples of the quaternary ammonium compound as a mixture of anoligomer with an oligomer or a monomer include an oligomer formed by thebonding between the alkenyl, the alkadiene, the alkatriene and/or theepoxy groups of quaternary ammonium compounds.

Preferably, the quaternary ammonium compound based on the formula (I) isglycidyl trimethylammonium, ethyl glycidyl dimethylammonium, diethylglycidyl methylammonium, or triethyl glycidyl ammonium, and isparticularly preferably glycidyl trimethylammonium.

Anion

The anion of the ionic liquid of the present invention is notparticularly limited as long as it is a conjugate Lewis baseconstituting the ionic liquid. The anion is preferably BF₄ ⁻, PF₆ ⁻,(CF₃SO₂)₂N⁻, a halide ion, a conjugate base of carboxylic acid, aconjugate base of sulfonic acid or a conjugate base of an inorganicacid. Among them, more preferred are BF₄ ⁻, PF₆ ⁻, and (CF₃SO₂)₂N⁻, andparticularly preferred is (CF₃SO₂)₂N⁻ because of their low meltingpoints and high heat resistance.

The anion may be commercial product or produced by a known method or amethod similar to a known method.

Ionic Liquid

The ionic liquid used in the present invention is not particularlylimited as long as it is a mixture of the above cation and anion.Preferably, the ionic liquid is glycidyl trimethylammoniumbis(trifluoromethanesulfonyl)imide, ethyl glycidyl dimethylammoniumbis(trifluoromethanesulfonyl)imide, diethyl glycidyl methylammoniumbis(trifluoromethanesulfonyl)imide, or triethyl glycidyl ammoniumbis(trifluoromethanesulfonyl)imide, and is particularly preferablyglycidyl trimethylammonium bis(trifluoromethanesulfonyl)imide.

The ionic liquid is produced by a known method, for example, by mixingthe anion and the cation.

The amount of the ionic liquid used is 1 to 50 vol %, preferably 5 to 35vol %, more preferably 7.5 to 25 vol %, and particularly preferably 10to 20 vol %, relative to the total volume of the composition.

Embedding Medium

The embedding medium used in the present invention comprises anepoxy-based resin, a methacrylate resin, or an unsaturated polyesterresin. For the preparation of the embedding medium of the presentinvention, already polymerized epoxy-based resin, methacrylate resin, orunsaturated polyester resin may be used. Alternatively, the epoxy-basedresin, methacrylate resin, or unsaturated polyester resin beforepolymerization may be used.

In view of strength, embedding performance and sectioning quality of theembedding medium of the present invention, the embedding mediumpreferably comprises an epoxy-based resin.

The amount of the epoxy-based resin, methacrylate resin, or unsaturatedpolyester resin used is 50 to 99 vol %, preferably 65 to 95 vol %, morepreferably 75 to 92.5 vol %, particularly preferably 80 to 90 vol %,relative to the total volume of the composition.

Epoxy-Based Resin

The epoxy-based resin can be prepared by, for example, mixing a monomerwith a polymerization initiator, a curing agent, and/or the like.

The monomer used to form the epoxy-based resin is not particularlylimited and may be an aliphatic epoxy monomer or an aromatic epoxymonomer. The monomer may be one kind or a mixture of two or more kinds.

Examples of the aliphatic epoxy monomer include3,4-epoxycyclohexylmethyl carboxylate,3,4-epoxy-6-methylcyclohexylmethyl carboxylate, dimer acid glycidylester, hexahydrophthalic acid glycidyl ester, diglycidyl ether,butanediol diglycidyl ether, hexahydrobisphenol A diglycidyl ether,polyethylene glycol diglycidyl ether, polypropylene glycol diglycidylether, neopentyl glycol diglycidyl ether, trimethylolpropane triglycidylether, glycerol triglycidyl ether, pentaerythritol triglycidyl ether,triglycidyl isocyanurate, tetraglycidyl-1,3-bisaminomethylhexane,dipentaerythritol hexaglycidyl ether, etc.

Examples of the aromatic monomer include a bisphenol A epoxy monomer, abisphenol F epoxy monomer, a bisphenol AD epoxy monomer, a bisphenol Sepoxy monomer, a novolac epoxy monomer, a biphenyl epoxy monomer, aglycidyl ether epoxy monomer, a glycidyl amine epoxy monomer, etc.

The monomer used to form the epoxy-based resin may be a commercialproduct or produced by a known method. Examples of the commercialproduct of the monomer used to form the epoxy-based resin includeAraldite CY-212, Epon 812, Epok 812 (all produced by ABBA company), etc.

Methacrylate Resin

The methacrylate resin can be prepared by, for example, mixing a monomerwith a polymerization initiator, a curing agent, and/or the like.

The monomer used to form the methacrylate resin is not particularlylimited but is preferably a mixture of a methacrylic acid ester, such asmethyl methacrylate and butyl methacrylate, with a styrene monomer.

Unsaturated Polyester Resin

The polyester resin can be prepared by, for example, mixing a monomerwith a polymerization initiator, a curing agent, and/or the like.

The monomer used to form the unsaturated polyester resin is notparticularly limited but is preferably a mixture of Rigolac (trade name)with a styrene monomer.

Curing Agent

The embedding medium comprising an epoxy resin used in the presentinvention preferably comprises a curing agent. The curing agent is notparticularly limited and examples thereof include carboxylic acidanhydrides, amines, sulfur-containing compounds, dicyandiamides, organichydrazides, etc. The curing agent may be one kind or a mixture of two ormore kinds.

The carboxylic acid anhydride is an anhydride of a carboxylic acidcontaining two or more carboxyl groups and is preferably an anhydride ofa carboxylic acid containing two carboxyl groups. The carboxylic acidanhydride may be an aliphatic carboxylic anhydride, a cyclic aliphaticcarboxylic anhydride, or an aromatic carboxylic anhydride.

Examples of the aliphatic carboxylic anhydride include acetic anhydride,maleic anhydride, propionic anhydride, succinic anhydride,acetylsuccinic anhydride, 3-dodecenyl succinic anhydride (DDSA), adipicanhydride, azelaic anhydride, citramalic anhydride, malonic anhydride,glutaric anhydride, citric anhydride, tartaric anhydride, oxoglutaricanhydride, pimelic anhydride, sebacic anhydride, itaconic anhydride,suberic anhydride, diglycol anhydride, etc.

Examples of the cyclic aliphatic carboxylic anhydride includehexahydrophthalic anhydride, cyclobutanedicarboxylic anhydride,cyclopentanedicarboxylic anhydride, norbornanedicarboxylic anhydride,hexahydrotrimellitic anhydride, tetrahydrophthalic anhydride,methyltetrahydrophthalic anhydride, tetrahydrophthalic anhydride, methylendo-methylenetetrahydrophthalic anhydride, chlorendic anhydride,methylhexahydro nadic anhydride, etc.

Examples of the aromatic carboxylic anhydride include phthalicanhydride, trimellitic anhydride, pyromellitic anhydride, mellophanicanhydride, naphthalic anhydride, etc.

Any of the hydrogen atoms or hydrocarbon groups of the above carboxylicanhydrides may be replaced or substituted with a substituent.

Examples of the amines include diethylenetriamine, triethylenetetramine,diethylamino propylamine, N-aminoethyl piperazine,bis(4-amino-3-methylcyclohexyl)methane, m-xylylenediamine,menthanediamine,3,9-bis(3-aminopropyl)-2,4,8,10-tetraoxaspiro[5.5]undecane, etc.

Examples of the sulfur-containing compounds include polysulfides,polymercaptans, etc.

Examples of the dicyandiamides include DICY-7 made by Yuka Shell EpoxyK.K., etc.

Examples of the organic hydrazides include adipic acid hydrazide,phthalic acid hydrazide, 7,11-octadecadiene-1,18-carbohydrazide,bisphenol A ether dicarboxylic acid hydrazide, etc.

The amount of the curing agent used is preferably 5 to 80 phr (parts perhundred parts of resin) relative to the embedding medium comprising anepoxy-based resin.

Optional Components

To the embedding medium of the present invention may be added a reactionaccelerator, a polymerization initiator, a color pigment, a filler, afiber, various additives, a solvent, a reactive diluent, etc. in anamount so as not to impair the effects of the invention.

Reaction Accelerator

The embedding medium comprising an epoxy resin used in the presentinvention preferably comprises a reaction accelerator, for example,tertiary amines such as triethylenediamine, benzyldimethylamine (BDMA),2-(dimethylaminomethyl)phenol (DMP-10) and2,4,6-tris(dimethylaminomethylphenol) (DMP-30); triphenylphosphine, etc.

Polymerization Initiator

The embedding medium comprising a methacrylate resin or an unsaturatedpolyester resin of the present invention preferably comprises apolymerization initiator. The polymerization initiator is notparticularly limited and may be a thermal polymerization initiator or aphoto polymerization initiator.

Examples of the photo polymerization initiator include radical reactioninitiators such as an alkylphenone-based radical initiator, anacylphosphine oxide-based radical initiator and a titanocene-basedradical initiator.

Examples of the thermal polymerization initiator include radicalreaction initiators such as benzoyl peroxide (BPO) andazobisisobutyronitrile (AIBN).

The embedding medium comprising an epoxy resin of the present inventionpreferably comprises a polymerization initiator. The polymerizationinitiator is not particularly limited and may be a photo polymerizationinitiator.

Examples of the photo polymerization initiator include cationicpolymerization initiators, for example, a salt containing a cation suchas diazonium, sulfonium, iodonium and oxonium and an anion such as PF₆⁻, SbF₆ ⁻, (C₆F₅)₄B⁻ and BF₄ ⁻; etc.

The amount of the polymerization initiator used is preferably 0.1 to 10phr (parts per hundred parts of resin) relative to the embedding mediumcomprising an epoxy-based resin, a methacrylate resin or an unsaturatedpolyester resin.

Preparation of Composition

The embedding resin composition for electron microscopy of the presentinvention can be obtained by mixing the ionic liquid with the embeddingmedium comprising an epoxy-based resin, a methacrylate resin or anunsaturated polyester resin.

The term “embedding resin composition for electron microscopy” hereinrefers to a liquid, paste or slurry composition used for embedding.Preferably, the embedding resin composition for electron microscopy is acomposition before curing.

Preferably, the embedding resin composition for electron microscopy ofthe present invention is prepared by mixing the ionic liquid with theembedding medium comprising an epoxy-based resin, a methacrylate resinor an unsaturated polyester resin and, after embedding of a sample inthe composition, polymerization and curing are performed. Polymerizationin the composition can be performed by, for example, exposing thecomposition to light or heating the composition at 40 to 80° C. for 24to 96 hours. Curing of the composition can be performed by, for example,heating the composition at normal temperature to 100° C. for 24 to 96hours.

Performance of Composition

Antistatic Performance

The embedding resin composition for electron microscopy of the presentinvention has antistatic performance. The embedding resin compositionfor electron microscopy of the present invention has antistaticperformance and can therefore prevent charging of a sample surface,thereby allowing detailed observations of the sample. In this way,sufficient resolution can be achieved.

The antistatic performance of the embedding resin composition forelectron microscopy of the present invention is, when expressed in termsof the surface resistance of the composition, 1.0×10¹⁵ Ω/sq or less,preferably 1.0×10¹⁴ Ω/sq or less, particularly preferably 7.5×10¹³ Ω/sqor less. When the surface resistance is in the above range, a samplesurface can be efficiently prevented from being charged, therebyallowing detailed observations of various non-conductive samples. Inaddition to this, sufficient resolution can be achieved.

Preferably, the antistatic performance is such that, when a sample isphotographed with a scanning electron microscope, no influence bycharging is observed on the image, i.e., no image defect or distortionis observed.

Performance as Embedding Resin Composition

The embedding resin composition for electron microscopy of the presentinvention satisfies performance generally required of embedding resincompositions, including sectioning quality and embedding performance.

The term “embedding performance” herein refers to the ability of, when asample is photographed with an electron microscope, providing an imagewith sufficient quality. The term “sectioning quality” herein refers tothe ability of providing sufficiently thin sections of an embeddedsample.

Use

The embedding resin composition for electron microscopy of the presentinvention can be used for embedding of a sample to be observed with anelectron microscope.

The sample to be embedded is not particularly limited as long as it canbe subjected to the embedding step described later, and the sample maybe a biological sample or a non-biological sample.

Biological Samples

Preferably, the biological sample is a cell or tissue of an animal orplant.

Examples of the plant include procaryotes, protists, fungi, algae,terrestrial plants, etc.

Examples of the procaryotes include bacteria such as Escherichia coli,lactic acid bacteria, Streptococcus pneumoniae, nitrite bacteria,nitrate bacteria, and sulfur bacteria; cyanobacteria such as greensulfur bacteria, purple sulfur bacteria, Oscillatoria, and Nostoc; etc.

Examples of the protists include protozoa, unicellular algae, etc.Examples of the protozoa include Rhizopoda such as amoebas; Ciliophorasuch as Paramecium; Mastigophora such as Trypanosoma; Sporozoa such asmalaria pathogens; etc. Examples of the unicellular algae includeEuglenida such as Euglena and Trachelomonas; Dinoflagellata such asCeratium and Noctiluca; Bacillariophyceae such as Pinnularia andNavicula; etc.

Examples of the fungi include Myxomycota, Eumycota, etc. Examples of theMyxomycota include Myxomycetes such as Stemonitis and Trichia; cellularslime molds such as Dictyostelium and Acrasis; etc. Examples of theEumycota include Oomycota such as Saprolegnia, Achlya, and Pythium;Zygomycetes such as Rhizopus, Mucor, and Zoopagales; Ascomycetes such asAspergillus, Penicillium, Neurospora, Peziza, and yeasts; Basidiomycetessuch as Tricholoma matsutake, Lentinula, Auricularia, and Flammulina;etc.

Examples of the algae include Red algae such as layer, agar-agar,Gloiopeltis, Meristotheca, and Batrachospermum; Brown algae such askelp, wakame, Hizikia, Nemacystus, Sargassum, and Padina; Green algaesuch as volvox, Chlamydomonas, chlorella, sea lettuce, and Acetabularia;stoneworts such as Chara and Nitella; etc.

Examples of the terrestrial plants include moss plants such asMarchantia, Conocephalum, Polytrichum, and Sphagnum; fern plants such aswhisk fern, clubmoss, common horsetail, scouring rush, bracken, andJapanese royal fern; gymnosperms such as cycads, ginkgo, Japanese redpine, Japanese cedar, and Japanese cypress; angiosperms such as riceplant, palm tree, and orchid; etc.

Examples of the animal include Porifera, Coelenterate, Platyhelminthes,Aschelminthes, Annelida, Mollusca, Arthropoda, Chaetognatha,Echinodermata, Vertebrata, etc.

Examples of the Porifera include Halichondria okadai, Haliclona,Euplectella, etc. Examples of the Coelenterate include sea anemones,corals, hydras, etc. Examples of the Platyhelminthes include planarians,tapeworms, Bipalium, etc. Examples of the Aschelminthes includeNemathelminthes such as nematodes, roundworms, pinworms, and hairworms;Trochelminthes such as Brachionus, Trichocerca, and Rotaria; etc.

Examples of the Annelida include earthworms, Tubifex, Neanthes, featherduster worms, leeches, etc. Examples of the Mollusca include pelecypodssuch as common orient clams and Corbicula; gastropods such as hornedturban shell and land snails; cephalopods such as octopuses and squids;etc. Examples of the Arthropoda include crustaceans such as water fleas,shrimps, and crabs; centipedes such as scolopendromorph centipede andThereuonema tuberculata; spiders such as Nephila clavata and mites;insects such as grasshoppers and beetles; etc. Examples of theChaetognatha include Flaccisagitta hexaptera etc. Examples of theEchinodermata include sea lilies, feather stars, sea cucumbers, seaurchins, star fishes, etc.

Examples of the Vertebrata include Protochordata such as sea squirts,Doliolida, and lancelets; Agnatha such as lampreys; cartilaginous fishessuch as sharks and rays; bony fishes such as carp and Pacific saury;amphibians such as frogs and newts; reptiles such as lizards, turtles,and snakes; birds such as pigeons, swallows, pheasants, sparrows, andchickens; mammals such as human, monkeys, lions, whales, rats, cattles,horses, and sheeps; etc.

Examples of the biological sample include a cell or tissue of an animalor plant.

Examples of the cell or tissue of an animal include epithelial tissuesuch as the epidermis of the skin, hair, nail, the inner wall of thedigestive tract, blood capillaries, and salivary gland; connectivetissue such as the dermis of the skin, tendon, blood, cartilage, bone,and fat; muscular tissue such as striated muscle, smooth muscle, andcardiac muscle; nervous tissue such as neurons; etc. and the cells ofthese tissues.

The examples of the cell or tissue of an animal further include thecells or tissue of organs, for example, digestive system organs such asoral cavity, salivary gland, esophagus, stomach, pancreas, gallbladder,liver, duodenum, small intestine, large intestine, appendix vermiformis,and rectum; circulatory system organs such as heart, aorta, inferiorvena cava, brachial artery, brachial vein, carotid artery, jugular vein,subclavian artery, and subclavian vein; respiratory system organs suchas trachea, bronchus, and lung; excretory system organs such as kidney,ureter, and urinary bladder; nervous system organs such as brain, spinalcord, and peripheral nerves; skeletal system organs such as cranialbone, vertebral column, pelvis, thigh bone, and shoulder blade; muscularsystem organs such as greater pectoral muscle, biceps brachii muscle,and frontal muscle; genital system organs such as gonad, oviduct, vasdeferens, womb, and placenta; endocrine system organs such as pituitarygland, thyroid gland, parathyroid gland, adrenal gland, pancreas, andgonad; sensory system organs such as eye, ear, nose, tongue, and skin;etc.

Examples of the cell or tissue of a plant include epidermal tissue,mechanical tissue, absorptive tissue, assimilation tissue, conductivetissue, storage tissue, aerenchyma, secretory tissue, etc. and the cellsof these tissues.

The examples of the cell or tissue of a plant further include the cellsor tissue of organs, for example, root, stem, leaves, seed, flower, etc.

Non-Biological Sample

The non-biological sample may be an organic substance or an inorganicsubstance. The non-biological sample may be a naturally occurring sampleor an artificially synthesized sample. Examples of the non-biologicalsample include resins, rubbers, synthetic resins, pigments, coatingmaterials, cosmetics, pharmaceutical drugs, ceramics, magnetic bodies,magnetic materials, semiconductors, metals, metal oxides, minerals,organic salts, inorganic salts, etc.

After a sample is harvested, the sample may be directly subjected toembedding or first dehydrated and then subjected to embedding. Thesample to be embedded may be a sample containing water.

The size of the sample is not particularly limited and the sample may bea powder. Examples of the powder sample include foods such as wheatflour, coffee, salt, sugar, starch, spices and seasonings;pharmaceutical drugs such as granules, cosmetics and powdered medicine;metal oxides such as alumina, iron oxide and tin oxide; metals, feeds,detergents, cosmetics, pigments, powdered paints, carbon toners,magnetic bodies, magnetic materials, cements, glass, abrasives, sand,semiconductors, ceramics, minerals, sintered bodies, gunpowders, etc.

Method for Observing Sample with Electron Microscope

Embedding Step

The method for observing a sample with an electron microscope of thepresent invention comprises the step of embedding a sample in the aboveembedding resin composition for electron microscopy.

The embedding step can be performed in accordance with a known method ora method similar to a known method except that the above composition isused. The embedding step comprises, for example, embedding a sample tobe observed in the embedding resin composition for electron microscopyof the present invention. In this step, the sample to be observed may bea non-conductive sample such as a biological sample, a non-biologicalsample, a powder sample, etc. When a sample containing water is used,the sample may be, for example, dehydrated in an ascending series ofalcohol, acetone, etc. and then embedded in the resin composition. Whena sample not containing water, for example, a non-biological sample or apowder sample is used, the sample may be directly embedded in the resincomposition or mixed with the resin composition so as to be embeddedtherein. When a biological sample is used, the sample may be, forexample, first fixed in glutaraldehyde etc. and then a part to beobserved is cut out and subjected to embedding.

According to the method of the present invention, since the embeddingresin composition for electron microscopy has antistatic performance,the surface structure of a sample can be sufficiently observed. Further,since the embedding resin composition for electron microscopy exhibitssatisfactory performance as an embedding resin composition, theembedding resin composition can be used for embedding of varioussamples.

Non-Stained Sample

The method for observing a sample with an electron microscope of thepresent invention can be used for observation of anon-stained sample.Anon-stained sample can provide image contrast which represents theelemental composition of the sample itself.

Staining Step The method for observing a sample with an electronmicroscope of the present invention can comprise the step of staining asample. The staining step enables detailed observations of the surfacestructure or internal structure of the sample. The staining step can beperformed on both biological samples and non-biological samples.

Staining can be performed by a known method. For example, the followingmethods can be employed:

1) en bloc staining: Osmic acid staining alone, tannic acid-osmic acidstaining (TaO method), or potassium ferrocyanide-osmicacid-thiocarbohydrazide-osmic acid multistaining (OTO method) isperformed and subsequently additional staining, such as uranyl acetatestaining, lead staining (Walton method), phosphotungstic acid staining,and potassium permanganate staining, is performed; or2) after preparation of the sample surface to be observed, directstaining of a sample surface with uranyl acetate solution is performed.

The staining step may be performed before or after the embedding step,but is preferably performed before the embedding step. This is becausestaining of a sample before embedding provides satisfactory stainingresults regardless of the resin used.

Preparation Step of Surface to be Observed

The method for observing of a sample with an electron microscope of thepresent invention preferably further comprises the step of thinsectioning a sample or exposing the surface of a sample. The thinsectioning step can be performed using a known device such as anultramicrotome and a polishing machine. In the thin sectioning step, asample is preferably thin sectioned into 50 to 100 nm thick slices.Exposure of the surface can be achieved by means of an ultramicrotomewith a diamond knife, a focused ion beam (FIB) apparatus, an ionpolishing machine, or a precision polishing machine, and the obtainedsurface can be observed with a scanning electron microscope.

The electron microscope used for the observation method of the presentinvention is not particularly limited and may be any of a transmissionelectron microscope (TEM), a scanning electron microscope (SEM) and ascanning transmission electron microscope (STEM). For the observation ofa sample surface, preferred are a SEM and a SEM having a built-in FIBapparatus.

EXAMPLES

The present invention will be illustrated below with reference toExamples, but the present invention is not limited thereto.

For observations of the surface of embedded biological samples, ascanning electron microscope (“S-800” produced by

Hitachi High-Technologies Corporation) was used. For observations of thesurface of embedded non-biological samples, a SEM having a built-in FIBapparatus (“Quanta FEG (FEI)” produced by Japan FEI Company) was used.

For thin sectioning, a microtome (“UltracutE” produced by Reichert AG(current Leica Microsystems GmbH)) was used.

The unspecified reagents used below are all commercial products.

Evaluation Method

Polymerizability was determined to be excellent in cases wherepolymerization of a composition occurred.

Sectioning quality was determined to be excellent in cases where asample embedded in a composition was successfully thin sectioned into 50nm thick slices.

Embedding performance was determined to be excellent in cases where thequality of a photograph of a sample taken by a SEM (scanning electronmicroscope) was comparable with that of a photograph of the sampleembedded in an embedding medium composition not containing the ionicliquid described later.

Antistatic performance in the observation of a biological sample wasdetermined to be excellent in cases where the surface resistance was1.0×10¹⁵ or less. Antistatic performance in the observation of anon-biological sample was determined to be excellent in cases where aphotograph of the sample taken by a scanning electron microscope showedno influence by charging, i.e., no image defect or distortion.

Observation of Biological Sample (Examples 1 to 3 and ComparativeExample 1) Example 1 Preparation of Epoxy-Based Resin

An epoxy-based resin was prepared according to Luft's formulation (6:4).EPON 812 resin kit produced by TABB Laboratories Equipment Ltd was used.An amount of 4.7 ml of EPON 812 (produced by Shell Chemicals Co.) as anepoxy monomer was mixed with 2.8 ml of methyl nadic anhydride (MNA) and2.5 ml of dodecenyl succinic anhydride (DDSA) as curing agents. To theresulting mixture, 0.15 ml of 2,4,6-tris(dimethylaminomethyl)phenol(DMP-30) as a polymerization accelerator was added to give anepoxy-based resin in a paste form.

Preparation of Embedding Resin Composition for Electron Microscopy

An amount of 4.00 ml of the above epoxy-based resin was mixed with 1.00ml of N-glycidyl trimethylammonium bis(trifluoromethanesulfonyl)imide(produced by Japan Carlit Co., Ltd.; hereinafter also referred to as“GTA-TFSI”) as an ionic liquid to give an embedding resin compositionfor electron microscopy in a paste form.

Embedding

Rat liver tissue was used as a sample to be observed. The liver tissuesample was fixed in 2% glutaraldehyde+2.5% formaldehyde solution. A cutout of the fixed sample was postfixed in 2% osmic acid/1% potassiumferrocyanide solution and en bloc staining was performed with 1% uranylacetate aqueous solution.

The sample to be observed was mixed with the embedding resin compositionand poured into a mold. The embedding resin composition was heated to atemperature range of 63 to 65° C. and this temperature was maintainedfor 48 hours so that the composition was polymerized and cured. Thecured embedding resin composition was cut and thin sectioned by means ofthe ultramicrotome with a diamond knife to produce a smooth surface onthe embedding resin composition. Thus the embedding resin composition tobe observed with the SEM was prepared. The composition was mounted onthe sample stage of the SEM with silver paste, and without furthertreatment such as conductive coating, the smooth surface of thecomposition was observed with the SEM.

Example 2

An experiment was conducted in the same manner as in Example 1 exceptthat the amount of the epoxy-based resin was 4.50 ml and that the amountof GTA-TFSI was 0.50 ml.

Example 3

An experiment was conducted in the same manner as in Example 1 exceptthat the amount of the epoxy-based resin was 4.75 ml and that the amountof GTA-TFSI was 0.25 ml.

Comparative Example 1

An experiment was conducted in the same manner as in Example 1 exceptthat the amount of the epoxy-based resin was 5.00 ml and that the amountof GTA-TFSI was 0 ml.

The results of Examples 1 to 3 and Comparative Example 1 are shown inTable 1.

TABLE 1 Embedding resin composition Epoxy-based Results Ionic liquidresin Antistatic performance Amount Mixing Amount Mixing Surface addedratio added ratio Sectioning Embedding resistance Evaluation (ml) (%)(ml) (%) Polymerizability quality performance (Ω/sq) results Example 11.00 20 4.00 80 Good Good Good 2.8 × 10¹³ Good Example 2 0.50 10 4.50 90Good Good Good 4.8 × 10¹³ Good Example 3 0.25 5 4.75 95 Good Good Good3.2 × 10¹⁴ Good Comparative 0 0 5.00 100 Good Good Good >10¹⁵ PoorExample 1

Evaluation Results

It was revealed that the embedding resin compositions of Examples 1 to 3had sufficient antistatic performance, while maintaining basicperformance required of an embedding resin composition, includingsectioning quality and embedding performance.

FIG. 1 shows a SEM secondary electron image observed in Example 1. Asshown in FIG. 1, no charging on the sample surface was observed and theshape of the sample surface could be sufficiently observed.

FIG. 2 shows a SEM secondary electron image observed in ComparativeExample 1. As shown in FIG. 2, charging on the sample surface wasobserved and the conditions of the surface could not be sufficientlyobserved.

Observation of Non-Biological Sample (Examples 4 and 5 and ComparativeExamples 2 and 3) Example 4 Preparation of Embedding Resin Compositionfor Electron Microscopy

An amount of 3.75 ml of the epoxy-based resin prepared in Example 1 wasmixed with 1.25 ml of GTA-TFSI as an ionic liquid to give an embeddingresin composition for electron microscopy in a paste form.

Embedding

A waste toner of a color copier was used as a sample to be observed. Anamount of 5 parts by weight of the sample to be observed was mixed with95 parts by weight of the embedding resin composition and poured into amold. The embedding resin composition was heated to a temperature rangeof 63 to 65° C. and this temperature was maintained for 48 hours so thatthe composition was polymerized and cured.

The cured embedding resin composition was cut and thin sectioned bymeans of the ultramicrotome with a diamond knife to produce a smoothsurface on the embedding resin composition. Thus the embedding resincomposition to be observed with the SEM was prepared. The compositionwas mounted on the sample stage of the SEM with silver paste, andwithout further treatment such as conductive coating, the smooth surfaceof the composition was observed with the SEM. The obtained secondaryelectron image and backscattered electron image are shown in FIGS. 3 and4, respectively.

Comparative Example 2

An experiment was conducted in the same manner as in Example 4 exceptthat a resin composition consisting of 5.00 ml of the epoxy-based resinand not containing the ionic liquid was used. The obtained secondaryelectron image and backscattered electron image are shown in FIGS. 5 and6, respectively.

Example 5

A plastic sticky note (with adhesive) was used as a sample to beobserved. The embedding resin composition in a paste form prepared inExample 1 was poured into a mold and the sample to be observed wasembedded. The embedding resin composition was heated at 63 to 65° C. for48 hours so that the composition was polymerized and cured. The surfaceof the cured embedding resin composition was cut and thin sectioned bymeans of the ultramicrotome to produce a smooth surface on the embeddingresin composition. Thus the embedding resin composition to be observedwith the SEM was prepared. The composition was mounted on the samplestage of the SEM with silver paste, and without further treatment suchas conductive coating, the smooth surface of the composition wasobserved with the SEM. The obtained secondary electron image andbackscattered electron image are shown in FIGS. 7 and 8, respectively.The backscattered electron image of the adhesive part of the plasticsticky note is shown in FIG. 9.

Comparative Example 3

An experiment was conducted in the same manner as in Example 5 exceptthat a resin composition consisting of 5.00 ml of the epoxy-based resinand not containing the ionic liquid was used. The obtained secondaryelectron image and backscattered electron image are shown in FIGS. 10and 11, respectively.

The results of Examples 4 and 5 and Comparative Examples 2 and 3 areshown in Table 2.

TABLE 2 Embedding resin composition Epoxy-based Ionic liquid resinAmount Mixing Amount Mixing Results added ratio added ratio SectioningEmbedding Antistatic (ml) (%) (ml) (%) Sample Polymerizability qualityperformance performance Example 4 1.25 25 3.75 75 Waste Good Good GoodGood toner Example 5 1.25 25 3.75 75 Sticky Good Good Good Good noteComparative 0 0 5 100 Waste Good Good Good Poor Example 2 tonerComparative 0 0 5 100 Sticky Good Good Good Poor Example 3 note

Evaluation Results

As is apparent from Table 2, the embedding resin compositions ofExamples 4 to 5 had sufficient antistatic performance, while maintainingbasic performance required of an embedding resin composition, includingsectioning quality and embedding performance.

As shown in the secondary electron image of Example 4 (see FIG. 3), whenthe embedding resin composition used was the mixture of the epoxy-basedresin and the ionic liquid, no charging on the sample surface wasobserved and the shape of the waste toner could be sufficientlyobserved. On the other hand, as shown in the secondary electron image ofComparative Example 2 (see FIG. 5), when the embedding resin compositionused was the composition consisting of the epoxy-based resin and notcontaining the ionic liquid, image defects and image distortion occurredand thus the shape of the waste toner could not sufficiently beobserved.

As shown in the backscattered electron image of Example 4 (see FIG. 4),clear compositional contrast of the toner could be obtained. On theother hand, as shown in the backscattered electron image of ComparativeExample 2 (see FIG. 6), due to strong charging on the sample, ameaningful image could not be obtained and thus the compositionalcontrast of the sample was not obtained.

As shown in the secondary electron image of Example 5 (see FIG. 7), whenthe embedding resin composition used was the mixture of the epoxy-basedresin and the ionic liquid, no charging on the sample surface wasobserved and the shape of the plastic sticky note could be sufficientlyobserved. On the other hand, as shown in the secondary electron image ofComparative Example 3 (see FIG. 10), when the embedding resincomposition used was the composition consisting of the epoxy-based resinand not containing the ionic liquid, image defects and image distortionoccurred and thus the shape of the plastic sticky note could notsufficiently be observed.

As shown in the backscattered electron image of Example 5 (see FIG. 8),when the embedding resin composition used was the mixture of theepoxy-based resin and the ionic liquid, no strong charging on the samplesurface was observed and thus the compositional contrast of the samplecould be obtained. The adhesive part of the sample could also be clearlyobserved (see FIG. 9). On the other hand, as shown in the backscatteredelectron image of Comparative Example 3 (see FIG. 11), strong noise dueto strong charging was observed and thus the sample could not besufficiently observed.

Example 6

The embedded waste toner of a color copier in Example 4 was used as asample to be observed. The sample surface was serially cut with FIB witha cutting thickness of 100 nm and the freshly cut smooth surface wasobserved with the SEM in the same manner as in Example 4. This cuttingand observation cycle was repeated 377 times to produce successiveimages of the cut surface of the embedded sample (observation area: 42μm×42 μm×38 μm, voxel size: 41.6 nm×41.6 nm×100 nm). The obtained imageswere processed with Avizo 6.3 software (VSG Inc., Bordeaux, France) toreconstruct a three-dimensional image and thus a three-dimensional imageof the waste toner particles was obtained. The obtained image is shownin FIG. 12.

Evaluation Results

As shown in the three-dimensional reconstruction image of Example 6 (seeFIG. 12), when the embedding resin composition used was the mixture ofthe epoxy-based resin and the ionic liquid, no influence due to chargingwas observed and thus the three-dimensional structure of the waste tonercould be observed in detail.

INDUSTRIAL APPLICABILITY

The embedding resin composition for electron microscopy of the presentinvention comprising an ionic liquid and an embedding medium comprisingan epoxy-based resin, a methacrylate resin or an unsaturated polyesterresin has antistatic performance and is therefore industrially useful.

1. An embedding resin composition for electron microscopy, thecomposition having antistatic performance and comprising an ionic liquidand an embedding medium comprising an epoxy-based resin, a methacrylateresin or an unsaturated polyester resin.
 2. The composition according toclaim 1, wherein the ionic liquid comprising a quaternary ammoniumcompound based on the formula (I):

(in the formula (I), R¹, R², R³ and R⁴ are independently a hydrogenatom, an alkyl group, an alkynyl group, an alkenyl group, an alkadienegroup, an alkatriene group, a cycloalkyl group or an aliphaticheterocyclic group and any of the hydrogen atoms of the groups may bereplaced with a substituent; at least one of R¹, R², R³ and R⁴ containsan alkenyl group, an alkadiene group, an alkatriene group or an epoxygroup; and R¹, R², R³ and R⁴ may bind to each other to form a ring) andan anion selected from the group consisting of BF₄ ⁻, PF₆ ⁻,(CF₃SO₂)₂N⁻, a halide ion, a conjugate base of carboxylic acid, aconjugate base of sulfonic acid and a conjugate base of an inorganicacid.
 3. The composition according to claim 2, wherein R¹ is an epoxygroup, a glycidyl group, or an alkenyl group of 2 to 10 carbon atoms. 4.The composition according to claim 2, wherein R², R³ and R⁴ areindependently an alkyl group of 1 to 6 carbon atoms.
 5. The compositionaccording to claim 2, wherein the quaternary ammonium compound is amonomer, an oligomer or a mixture thereof.
 6. The composition accordingto claim 2, wherein the anion is (CF₃SO₂)₂N⁻.
 7. The compositionaccording to claim 1, wherein the amount of the ionic liquid is 5 to 35vol % relative to the total volume of the composition.
 8. A method forobserving a sample with an electron microscope, the method comprisingthe step of embedding a sample in the composition according to claim 1.9. The observation method according to claim 8, wherein the methodcomprises the step of staining a sample and the staining step isperformed before the embedding step.
 10. The observation methodaccording to claim 8, wherein the electron microscope is a scanningelectron microscope.
 11. The observation method according to claim 8,wherein the sample is a biological sample.
 12. The observation methodaccording to claim 11, wherein the biological sample is a cell or tissueof a plant or animal.
 13. The observation method according to claim 8,wherein the sample is a non-biological sample.
 14. The observationmethod according to claim 13, wherein the non-biological sample is aresin, a rubber, a synthetic resin, a pigment, a coating material, acosmetic, a pharmaceutical drug, a ceramic, a magnetic body, a magneticmaterial, a semiconductor, a metal, a metal oxide, a mineral, an organicsalt, or an inorganic salt.
 15. The observation method according toclaim 8, wherein the sample is a powder.